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Experimental Therapeutics, Preclinical Pharmacology |
Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [S. Y., R. S. H., H. S., B. K., C. D. B., J. J. K., I. J. F.], and Oncology Research , Novartis, Ltd., CH-4002 Basel, Switzerland [J. W.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Among the possible targets for PE treatment is VEGF, also called VPF (5) , an important multifunctional cytokine that promotes developmental, physiological, and pathological neovascularization (6, 7, 8) . VEGF/VPF consists of at least four isoforms (VEGF121, VEGF165, VEGF189, and VEGF206), arising through alternate splicing of mRNA from a single gene (6) . It can be produced by various cell types, including many tumor cells and activated macrophages (6) . VEGF/VPF has been shown to stimulate the proliferation and migration of endothelial cells and to induce the expression of metalloproteinases and plasminogen activity by these cells (6 , 9, 10, 11, 12) . The cytokine is also a powerful inducer of vascular hyperpermeability. Through this property, the molecule plays a central role in ascites fluid formation by murine tumors and human ovarian cancer cell lines in animal models (13, 14, 15, 16, 17) .
Recently, we developed a model for PE by human lung adenocarcinoma cells (PC14 and its highly metastatic variant, PC14PE6) in nude and severe combined immunodeficiency mice (18) and clarified the role of VEGF/VPF in PE formation. The i.v. injection of PC14 and PC14PE6 cells, expressing high levels of VEGF/VPF, produced lung lesions that in turn produced large-volume PE in mice. On the other hand, i.v. or intrathoracic injection of H226 cells (VEGF/VPF low-expressing human lung squamous cell carcinoma) produced lung lesions without detectable PE. Interestingly, VEGF/VPF gene transfection into H226 cells resulted in induction of PE when tumor cells were injected intrathoracicly.4 This evidence suggests that VEGF/VPF plays a crucial role in PE formation by human non-small cell carcinoma cells.
Two VEGF/VPF receptors have been identified: Flt-1 and the Flk-1/kinase insert domain-containing receptor (KDR), are high-affinity VEGF/VPF receptors with an extracellular domain containing seven immunoglobulin-like domains and a split tyrosine kinase intracellular domain (6) . Flk-1 has 85% homology with the human homologue, KDR. Both Flt-1 and Flk-1/KDR have been shown to be important regulatory systems for vasculogenesis and physiological angiogenesis (19, 20, 21, 22, 23, 24) . However, the interaction of VEGF/VPF with Flk-1/KDR is thought to be the more important interaction for tumor angiogenesis because it is essential for induction of the full spectrum of VEGF/VPF functions (6) . In fact, many compounds and molecules developed to block VEGF/VPF activities mediated by Flk-1/KDR have been shown to have antiangiogenic activity in animal models (25, 26, 27) .
One such molecule is an inhibitor of tyrosine kinase phosphorylation of Flk-1/KDR and Flt-1, called PTK 787 (28) . PTK 787 directly inhibits phosphorylation of the VEGF/VPF receptor tyrosine kinases and suppresses angiogenesis induced by VEGF/VPF. At slightly higher doses, it also inhibits PDGF receptor tyrosine kinase phosphorylation (28) . It can be given p.o., is well tolerated, and has been demonstrated to inhibit the growth of several carcinomas in nude mice (28) .
In this study, we examined the therapeutic efficiency of PTK 787 against malignant PE caused by human lung adenocarcinoma cells (PC14PE6) established in nude mice. PTK 787 specifically inhibited VEGF/VPF-induced proliferation of human dermal endothelial cells and had no direct effect against PC14PE6 cells. We found that oral feeding with PTK 787 suppressed the formation of malignant PE by inhibiting vascular permeability. These findings suggest that therapy with the VEGF/VPF receptor tyrosine kinase phosphorylation inhibitor PTK 787 is worthy of study in clinical trials.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents.
rh VEGF165, rhbFGF, and antihuman VEGF polyclonal antibody were
purchased from R&D Systems (Minneapolis, MN). PTK 787/ZK232394 was
discovered and synthesized in the Department of Oncology Research,
Novartis Pharmaceuticals (Basel, Switzerland) and was profiled in
collaboration with the Institute of Molecular Medicine (Tumor Biology
Center, Freiburg, Germany), as well as the Oncology Research
Laboratories of Schering AG (Berlin, Germany). The studies described in
this report were performed with either a dihydrochloride or succinate
salt. For in vitro assays, a stock solution of 10
mM of PTK787/ZK 222584 was prepared in DMSO. This
was diluted further in buffer or medium so that the concentration of
DMSO in assay systems did not exceed 0.1%. For in vivo
studies, the vehicle for the dihydrochloride salt was distilled water.
The succinate salt was suspended in vehicle containing 5% DMSO and
0.5% Tween 80 in distilled water (28)
. The PDGF receptor
tyrosine kinase phosphorylation inhibitor CGP57148 (28
, 29)
was from Novartis.
Cell Proliferation Assay.
HDMECs (5 x 103/well) plated in triplicate
in 96-well plates precoated with 1.5% gelatin were incubated overnight
in supplemented M131 medium. PC14PE6 cells (2 x
103/well) plated in triplicate in 96-well plates
were incubated in MEM containing 5% FBS. The cells were then washed
and incubated for 72 h with test samples in fresh MEM containing
5% FBS. The proliferative activity was determined by the
3-(4,4-dimethylthiazol-2-yl)-2.5-diphenyl-tetrazolium bromide assay
using an MR-5000 96-well microtiter plate reader set at 570 nm
(12)
.
Animals.
Male athymic BALB/c nude mice were purchased from the Animal Production
Area of the National Cancer Institute, Frederick Cancer Research
Facility (Frederick, MD). The mice were housed in laminar flow cabinets
under specific pathogen-free conditions and used at 6–8 weeks of age.
Animals were maintained in facilities approved by the American
Association for Accreditation of Laboratory Animal Care and in
accordance with current regulations and standards of the United States
Department of Agriculture, Department of Health and Human Services, and
NIH.
Model for Lung Metastasis and PE.
Cultured PC14PE6 cells were harvested by pipetting. The cells were
washed twice and resuspended in Ca2+- and
Mg2+-free HBSS. Cell viability was determined by
a trypan blue exclusion test, and only single-cell suspensions of
>90% viability were used. Tumor cells (1 x
106/300 µl of HBSS) were injected into the
lateral tail vein of unanesthetized nude mice (18)
. After
the indicated periods, mice were euthanized by methoxyflurane, the
subclavian artery was severed, and blood was harvested. The chest wall
was then cut carefully with a pair of scissors, PE was harvested using
a 1-ml syringe, and the volume of PE was measured using the syringe.
The blood and PE harvested were centrifuged for 20 min (100 x
g) at 4°C. The serum and supernatant of PE were stored at
-70°C until the ELISA was performed. The lungs were fixed in
Bouin’s solution, and the number of lung lesions was determined with
the aid of a dissecting microscope.
Although this model does not entirely mimic all steps for PE formation in humans, this is one model in which PE is reproducibly developed, as reported previously (18) .
Assays of VEGF/VPF, bFGF, and IL-8 Protein Levels.
Levels of VEGF/VPF, bFGF, and IL-8 protein in culture supernatants,
mouse serum, and PE were determined using ELISA kits according to the
manufacturer’s instructions (R&D Systems, Minneapolis, MN).
Oligonucleotide Probes.
We designed antisense oligonucleotide DNA probes complementary to the
mRNA transcripts of the VEGF/VPF genes based on published
reports of the cDNA sequence (15)
TGGTGATGTTGGACTCCTCAGTGGGCU, 57.7% guanosine-cytosine (GC) content.
The specificity of the oligonucleotide sequence was initially
determined by a GenBank European Molecular Biology Library database
search with the use of the Genetics Computer Group sequence analysis
program (Madison, WI) based on the FastA algorithm. A
poly(dT)20 oligonucleotide was used to verify the
integrity and lack of degradation of mRNA in each sample. All DNA
probes were synthesized with six biotin molecules (hyperbiotinylated)
at the 3' end via direct coupling using standard phosphoramidite
chemistry (Research Genetics, Huntsville, AL).
ISH.
ISH was performed as described previously (30)
.
Tissue sections (4 µm) of formalin-fixed, paraffin-embedded specimens
were mounted on silane-treated ProbeOn slides (Fisher Scientific Co.).
The slides were placed in the Microprobe slide holder (Fisher
Scientific Co.), dewaxed, and rehydrated with Autodewaxer and
Autoalcohol (Research Genetics), digested with pepsin, and then
hybridized by use of the Microprobe manual staining system (Fisher
Scientific Co.). The probes were hybridized for 45 min at 45°C, and
the samples were then washed three times for 2 min each time with 2x
SSC (1x SSC = 0.15 M NaCl, 0.15 M
sodium citrate) at 45°C. RNase-free water was used to make up Tris
buffer and 2x SSC solutions. The samples were then incubated with
alkaline phosphate-labeled avidin for 30 min at 45°C, rinsed in 50
nM Tris buffer (pH 7.6), rinsed with alkaline phosphate
enhancer for 1 min, and incubated with a chromogen substrate for 20 min
at 45°C. Additional incubation with fresh chromogen was done if it
was necessary to enhance a weak reaction. A positive enzymatic reaction
in this assay stained red. Known positive controls were used in each
hybridization reaction. Controls for endogenous alkaline phosphate
included treatment of the sample in the absence of the biotinylated
probe and use of chromogen alone.
Histology and IHC.
The lungs of nude mice were harvested at autopsy, cut into 5-mm
thickness, and placed in either buffered 10% formalin solution or OCT
compound (Miles Laboratories, Elkhart, IN) to be snap-frozen in liquid
nitrogen. For VEGF/VPF staining, tissue sections (4 µm thick) of
formalin-fixed, paraffin-embedded specimens were deparaffinized in
xylene, rehydrated in graded alcohol, transferred to PBS, and treated
with pepsin for 20 min at room temperature. For CD31 staining, frozen
tissue sections (8 µm thick) were fixed with cold acetone. The slides
were rinsed twice with PBS, and endogenous peroxidase was blocked by
use of 3% hydrogen peroxide in PBS for 12 min. The samples were washed
three times with PBS and incubated for 10 min at room temperature with
a protein-blocking solution consisting of PBS (pH 7.5) containing 5%
normal horse serum and 1% normal goat serum. Excess blocking solution
was drained, and the samples were incubated for 18 h at 4°C with
a 1:400 dilution of rabbit polyclonal anti-VEGF/VPF antibody (Santa
Cruz Biotechnology, Santa Cruz, CA) or a 1:100 dilution of monoclonal
rat anti-CD31 antibody (PharMingen, San Diego, CA). The samples were
then rinsed four times with PBS and incubated for 60 min at room
temperature with the appropriate dilution of peroxidase-conjugated
antirabbit IgG or antirat IgG. The slides were rinsed with PBS and
incubated for 5 min with diaminobenzidine (Research Genetics,
Huntsville, AL). The sections were then washed three times with
distilled water, counterstained with Gill’s hematoxylin, washed once
with distilled water and once with PBS, and rinsed again with distilled
water. The slides were mounted with a Universal mount (Research
Genetics) and examined using a bright-field microscope. A positive
reaction was indicated by a reddish-brown precipitate in the cytoplasm.
Sections (4 µm thick) of formalin-fixed, paraffin-embedded tumors
were also stained with H&E for routine histological examination.
Vascular Density.
Blood vessels in solid tumors growing in the lungs of nude mice were
counted under light microscope after immune staining of sections with
anti-CD31 antibody. Areas containing the highest number of capillaries
and small venules were identified by scanning the tumor sections at low
power (x40). After the areas of high vascular density were identified,
individual vessels were counted in x100 fields [x10 objective and
x10 ocular (0.145 mm2)/field)]. On the basis of
criteria described by Weidner et al. (31)
,
observation of a vessel lumen was not required for a structure to be
classified as a vessel.
Permeability Assay (Miles Assay).
The Miles assay uses intradermal injection of test substances and
intravascular injection of Evans blue dye (which binds to endogenous
serum albumin) as a tracer to assay permeability in peripheral vessels.
The assay was performed essentially as described (32
, 33)
with minor modification. To reduce individual variation, nude mice
without downy hair were carefully chosen, and each mouse was kept
separately during the assay. Nude mice were injected i.v. with 200 µl
of 0.5% Evans blue dye (Sigma Chemical Co., St. Louis, MO). Ten min
later, 50-µl of samples were injected intradermally in rows on the
dorsal skin. Thirty min after the injection with samples, the mice were
killed, and the skin was removed. Wheals (5 mm in diameter) were
resected and incubated in 500 µl of formamide at 37°C for 48 h
to extract Evans blue dye. The absorbance of the extracts was
read at 630 nm in a spectrophotometer.
Statistical Analysis.
The significance of differences in data of in vitro
experiments, the Miles assay, and vessel density were analyzed using
Student’s t test (two-tailed). The remaining in
vivo data were analyzed using the Mann-Whitney U test
or 
2 test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Addition of rhVEGF/VPF or rhbFGF to the medium significantly stimulated
HDMEC proliferation, suggesting that HDMECs express receptors for
VEGF/VPF and bFGF, as is the case with most endothelial cell lines.
Under these experimental conditions, PTK 787 inhibited proliferation of
HDMECs stimulated by rhVEGF165 but not rhbFGF, indicating the
specificity of PTK 787 to VEGF/VPF receptors (
). In
contrast, PTK 787 did not affect proliferation of PC14PE6 cells,
irrespective of the presence of rhVEGF165 or rhbFGF (Fig. 1B
). Moreover, a PDGF-receptor tyrosine kinase
phosphorylation inhibitor, CGP57148, used as a control did not affect
the proliferation of HDMECs or PC14PE6 cells, irrespective of the
presence of rhVEGF165 or rhbFGF (Fig. 1, C and D
).
|
. Treatment with 100 mg/kg PTK 787
inhibited lung weight, although the reduction in the number of lung
metastases did not reach significance. In addition, PE formation (both
incidence and volume) was remarkably inhibited in this group. In
experiment 2, we examined the effect of a lower dose of PTK 787 (50
mg/kg) on formation of PE and lung metastasis. All mice of the control
group developed lung metastasis and PE, and treatment with 10 mg/kg PTK
787 again had no effect, consistent with the results in experiment 1.
The number of lung metastases or lung weight was not inhibited
significantly by treatment with 50 mg/kg PTK 787; however, this
treatment remarkably inhibited PE formation (both incidence and
volume). In experiment 3, the effect of oral treatment with the PDGF
receptor tyrosine kinase inhibitor CGP57148 was examined using the same
model. Although therapy with CGP57148 was started earlier (on day 7)
than therapy with PTK 787, it had no significant therapeutic effects on
the formation of PE or lung metastasis. These results suggest that PTK
787 specifically inhibits PE formation produced by PC14PE6 cells. It
also inhibited lung weight (total tumor volume of lung metastases) at
the highest dose (100 mg/kg) tested. On the basis of these results, we
chose to use 50 mg/kg PTK 787 in the following experiments.
|
, treatment with PTK 787 did not
suppress the levels of VEGF/VPF, bFGF, or IL-8 in the serum or PE.
Next, we examined the effect of PTK 787 treatment on VEGF/VPF
expression in lung tumors by ISH and IHC. Treatment with PTK 787 did
not affect VEGF/VPF expression in lung metastasis produced by PC14PE6
cells (Fig. 2)
. We also found that
production of bFGF or IL-8 in lung metastases was not affected by
treatment with PTK 787 in vivo (data not shown). However,
angiogenesis (one of the two major phenomena induced by VEGF/VPF)
quantitated in lung lesions of PTK 787-treated mice was inhibited
compared with that of control mice (number of CD31 positive cells x100
field; 34 ± 7 versus 52 ± 5; P < 0.05). This effect, in the absence of decreased cytokine levels,
suggests that treatment with PTK 787 might exert its effect on
endothelial cells as opposed to tumor cells.
|
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, rhVEGF165 and PE
(containing 30 ng/ml VEGF/VPF) caused by PC14PE6 cells induced vascular
hyperpermeability in control and CGP57148-treated mice. However,
neither rhVEGF165 nor PE produced in vivo by PC14PE6 cells
significantly enhanced permeability in mice treated with PTK 787. These
findings indicate that treatment with PTK 787 can inhibit induction of
hyperpermeability caused by VEGF/VPF, presumably by blocking VEGF/VPF
receptor function. We further explored how many treatments with PTK 787
were necessary for inhibition of vascular permeability. Results shown
in Fig. 3B
indicate that a 2–3-day treatment with PTK 787
was enough to inhibit the induction of vascular permeability in this
assay.
|
). These findings further suggest that
treatment with PTK 787 inhibited vascular permeability of PE-bearing
mice.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The interaction of VEGF/VPF and its receptors (Flt-1 and Flk-1/KDR) has been shown to play an important role in angiogenesis of malignant diseases (25, 26, 27) . Therefore, VEGF/VPF and its receptors (especially Flk-1/KDR) represent ideal targets for antiangiogenesis therapy. PTK 787 is a selective inhibitor of VEGF/VPF receptor tyrosine kinase phosphorylation and has been shown to inhibit VEGF/VPF-mediated responses in vitro and in vivo (28) .
In this study, we found that PTK 787 did not affect the in vitro proliferation of PC14PE6 cells, whereas it specifically inhibited the proliferation of HDMECs (presumably expressing VEGF/VPF receptors) stimulated with VEGF/VPF. In addition, treatment with PTK 787 inhibited vascularization in lung tumors produced by PC14PE6 cells, although it did not affect VEGF/VPF expression in tumors formed by PC14PE6 cells. These findings strongly suggest that treatment with PTK 787 directly inhibits endothelial cell function but not tumor cell function.
The two major functions of VEGF/VPF are induction of angiogenesis and
vascular hyperpermeability, both of which are thought to be mediated
mainly by Flk-1/KDR (6
, 33)
. In this study, we found that
treatment with PTK 787 inhibited the two major functions of VEGF/VPF.
Treatment with the highest dose (100 mg/kg) of PTK 787 inhibited
vascularization in the lung metastasis and it inhibited total tumor
volume, represented by lung weight. However, this treatment did not
significantly reduce the number of lung metastases, and there was no
significant correlation between the number of lung metastases and the
volume of PE (Fig. 5)
. This is not
unexpected because angiogenesis is not necessary for small tumors
(<1–2 mm in diameter; Ref. 34
). On the other hand, skin
vascular permeability induced by rhVEGF/VPF or PE (containing VEGF/VPF)
was significantly inhibited by treatment of the mice with PTK 787 as
shown in Fig. 3
. Moreover, treatment with 50 mg/kg PTK 787 for 6 days
significantly inhibited the vascular permeability of PE-bearing mice
(Fig. 4)
. Collectively, these findings suggest that a primary mechanism
by which treatment with PTK 787 inhibited PE was suppression of
vascular permeability.
|
Recently, various compounds that inhibit the function of VEGF/VPF and VEGF/VPF receptors, including humanized neutralization antibody for VEGF/VPF (38) , dominant-negative VEGF/VPF (39) , soluble VEGF/VPF receptors (26 , 40) , and low molecular weight compounds that inhibit VEGF/VPF receptor tyrosine kinases (27) have been developed, and their antiangiogenic activities have been demonstrated. The main advantages of PTK 787 over these compounds are as follows: (a) PTK 787 is a smaller compound with a low molecular weight and is easier to synthesize; (b) PTK 787 can be administered p.o. and hence may improve patient compliance. PTK 787 was very effective in this animal model. The drug administered daily for at least 35 days did not produce undesirable side effects. To confirm its efficacy, it will be necessary to evaluate the ability of PTK 787 to control malignant PE with a high level of VEGF/VPF in lung cancer patients receiving long-term treatment.
In summary, we demonstrate that the p.o.-administered VEGF/VPF receptor tyrosine kinase inhibitor PTK 787 inhibits the formation of malignant PE by human lung adenocarcinoma cells, through the inhibition of vascular permeability. Therefore, PTK 787 could be useful for the control of malignant PE in lung cancer patients, and clinical trials are warranted.
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FOOTNOTES |
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1 Supported in part by Cancer Center Support Core
Grant CA16672 and Grant R35-CA42107 (to I. J. F.) from the National
Cancer Institute, NIH. 
2 To whom requests for reprints should be
addressed, at the Department of Cancer Biology, Box 173, The University
of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard,
Houston, TX 77030. Phone: (713) 792-8577; Fax: (713) 792-8747;
E-mail: ifidler{at}notes.mdacc.tmc.edu
3 The abbreviations used are: PE, pleural
effusion; VEGF/VPF, vascular endothelial growth factor/vascular
permeability factor; Flt-1, fms-like tyrosine kinase; Flk-1, fetal
liver kinase; KDR, kinase insert domain-containing receptor; FBS, fetal
bovine serum; bFGF, basic fibroblast growth factor; PDGF,
platelet-derived growth factor; IL, interleukin; rh, recombinant human;
ISH, in situ hybridization; IHC, immunohistochemistry;
HDMEC, human dermal microvascular endothelial cell; PTK 787/ZK232394,
1-[4-chloroanilino]-4-[pyridylmethyl] phthalazine
dihydrochloride.
4 S. Yano, H. Shinohara, R. S. Herbst, H.
Kuniyasu, C. D. Bucana, L. M. Ellis, and I. J. Fidler.
Production of malignant pleural effusions is dependent on invasion of
the pleura and expression of vascular endothelial growth
factor/vascular permeability factor by human lung cancer cells,
submitted for publication.
Received 10/ 7/99; revised 12/ 9/99; accepted 12/13/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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